The role of cofilin in the migration of metastatic mammary cancer cells
Sidani, Mazen Moustafa
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Cofilin is a key player in the regulation of actin dynamics because of its ability to sever F-actin and increase the number of filament ends for polymerization and depolymerization. We have investigated the effects of inhibition of cofilin expression, using siRNA, in an apolar, randomly-moving, amoeboid metastatic mammary adenocarcinoma (MTLn3) cells. Cofilin knockdown MTLn3 (CF KD) cells extended protrusions from only one pole, and exhibited an elongated phenotype. CF KD cells showed an increase in the formation of prominent F-actin structures and aggregates, and total F-actin levels. Expressing a human cofilin plasmid (DeltaHCOF) restored the normal elongated phenotype of the CF KD cells.;Analysis of the constitutive motility of CF KD cells showed that they exhibited directional motility as compared to the random-walking behavior of control cells. CF KD cells showed a significant increase in their net and total path lengths, directionality and in the persistence of their movement. CF KD cells exhibited decrease in the degree of their direction change and in turning frequency. Cells rescued with the DeltaHCOF plasmid showed similar motility behavior to control cells. MTLn3 cells overexpressing full-length Lim Kinase, which phosphorylates and inactivates cofilin, showed an elongated phenotype along with an increase in their directionality. CF KD in MTC cells, a non-metastatic mammary adenocarcinoma cell line with a characteristic rectilinear type of motility, led to the formation of multipolar lamellipodia and to a decrease in the directionality of migration.;Kymography analysis showed that the protrusions in CF KD cells were more persistent, less frequent, but concentrated in just a few areas of the cell membrane, unlike the equally distributed protrusions in control cells. CF KD also suppressed the EGF-induced F-actin assembly at the leading edge, and inhibited the corresponding protrusive activity. CF KD cells were also less chemotactic and exhibited different sensitivity regions along the cell periphery, compared to control cells, which protrude equally when stimulated at different sites around their membrane. Finally, CF KD cells showed re-localization of the Arp2/3 complex to one pole of the cell suggesting that it contributes to the directionality and decreased turning frequency seen in the CF KD cells.